ALBERT

Description: We and others have shown that the tyrosine kinase inhibitor imatinib (STI571, Gleevec®) inhibits T-cell proliferation and activation at concentrations achieved in vivo. At 10μM, imatinib inhibited T-cell receptor (TCR)-mediated proliferation of purified peripheral blood T-cells almost completely. Up-regulation of the activation markers CD25 and CD69 at 24h in response to TCR cross-linking was suppressed by imatinib at a mean IC50 of 5.4μM and 7.3μM, respectively and IL-2 production was also severely impaired. However, these assays may not fully reflect the response to clinical relevant antigens. Therefore, we chose to investigate the antigen-triggered proliferation of memory CD8+ T-cells specific for immunodominant CMV and EBV HLA-A2 peptide epitopes. We used HLA-peptide tetramers to identify healthy blood donors with detectable CMV- or EBV-specific CD8+ T-cell populations. Purified T-cells from these donors were then stimulated with the CMV peptide pp65495–503 or the EBV peptide BMFLI259–267. Antigen-induced proliferation was measured by dilution of the vital dye CFSE over a period of 4 or 8 days. The magnitude of the virusspecific CD8+ T-cell population ranged from 0.5 % to 7.1% of CD8+ T-cells for CMV and from 0.05% to 0.35% of CD8+ T-cells for EBV. Antigen-specific CD8+ T-cells from all 10 donors studied proliferated in response to the CMV peptide. In 8 from 10 donors, imatinib reduced CMV peptide induced proliferation. With increasing imatinib concentrations (range: 5 – 10μM), we observed dose dependent reduction of both the number of cells undergoing cell division and the average number of divisions completed per cell. Comparable inhibition of specific T-cell proliferation in response to the EBV-derived peptide was observed in two donors. Immunoblots demonstrated that imatinib substantially reduced tyrosine phosphorylation of ZAP70 and LAT in response to TCR-mediated activation in Jurkat T-cells. Sequence comparisons of all 90 tyrosine kinase genes in the human genome for homology in the ATP binding pocket identified Lck, which is required for ZAP70 activation, as a likely target for imatinib. Our results indicate that imatinib may interfere with clinically important T-cell effector functions. As concentrations sufficient for half-maximal inhibition of TCR signalling are achieved in vivo, imatinib could increase the risk of opportunistic infections and impact on GVH and GVL reactions post-transplantation especially when used in conjuction with other immunosuppressive agents. Therefore, close monitoring of patients on imatinib for CMV reactivation or EBV-induced lymphoproliferative diseases, especially in stem cell transplant recipients, appears warranted.

Description: We recently completed a longitudinal study over 12 months on T-cell immune reconstitution after total body irradiation and autologous transplantation of peripheral blood progenitor cells in 3–5 year old rhesus macaques. The focus of the study was to evaluate the source of T-cell recovery using three different sources of cells for transplantation. The starting number of CD34+ cells contained within the graft was adjusted to be the same for each of the three groups: unselected peripheral blood progenitor cells (PBPC) (n=3) vs. CD34+ selected mobilized PBPC (n=4) vs. CD34+ selected cells cultured for 4 days in vitro (Flt-3L/MDGF/SCF/retronectin) and retrovirally-transduced (n=3). Peripheral blood and lymph nodes were collected for phenotypic and TREC analysis. There was a trend for animals receiving cultured and transduced CD34+ cells to attain higher absolute naive and memory CD4+ T-cell, CD20+ B-cell and CD16+ NK-cell numbers in the first months after transplantation compared to the two other groups. There were no differences for numbers of CD8+ T-cells, monocytes or dendritic cells. The absolute TREC amount per μl blood of the CD4+ T-cells in the group that received selected-transduced cells was significantly higher than in the group that received unselected cells (p=0.0166) and for CD8+ cells significantly higher than for selected cells (p=0.0464). There was significantly less peripheral T-cell expansion as measured by Ki-67 expression in the selected-transduced group compared to the two other groups: At 1 month after transplantation the mean Ki-67 expression level for CD8+ cells in peripheral blood was 33.9% in the selected vs. 23.3% in the unselected vs. 8% in the selected-transduced group. Histology at 12 months revealed striking differences in the thymus between the 3 groups, while other organs (lymph nodes, spleen, tonsils and Peyer’s patches) showed no remarkable differences. In the CD34+ selected-transduced group the thymus showed preserved lobular architecture with well defined cortical and medullary areas, compared to atrophy with fat replacement, decreased thickness of the cortex, and cystic changes of the thymic epithelium in the CD34+ selected and unselected groups. The degree of atrophy was more pronounced in the latter group. This study demonstrates an enhanced ability of in vitro expanded and retrovirally transduced cells to repopulate the thymus compared to non-manipulated CD34+ selected cells. In vitro expansion and transduction may promote development of T-cell and NK-cells as has been described earlier for fetal thymic organ culture of cord blood CD34+ cells as well as in the SCID-hu mouse model. Our observation of an acceleration of T-cell and NK cell immune reconstitution following in vitro culture contrasts with limited prior clinical trial experiences describing poor repopulating potential of cultured progenitor cells. Our data suggest that in vitro culture and retroviral gene transfer per se does not induce an intrinsic differentation defect. Future studies are planned to examine mechanisms behind the improved repopulation ability of selected-transduced progenitor cells, which has important implications for gene therapy trials, and suggests that cultured cells may be useful for a number of clinical applications.

Description: Allogeneic hematopoietic stem cell transplantation (HSCT) has advanced to a common procedure for treating also older patients with malignancies and immunodeficiency disorders by redirecting the immune system. Unfortunately, cure is often hampered by relapse of the underlying disease, graft-versus-host disease, or severe opportunistic infections, which account for the majority of deaths after HSCT. Enhancing immune reconstitution is therefore an area of intensive research. An increasing variety of approaches has been explored preclinically and clinically: the application of cytokines, keratinocyte growth factor, growth hormone, cytotoxic lymphocytes, and mesenchymal stem cells or the blockade of sex hormones. New developments of allogeneic HSCT, for example, umbilical cord blood or haploidentical graft preparations leading to prolonged immunodeficiency, have further increased the need to improve immune reconstitution. Although a slow T-cell reconstitution is regarded as primarily responsible for deleterious infections with viruses and fungi, graft-versus-host disease, and relapse, the importance of innate immune cells for disease and infection control is currently being reevaluated. The groundwork has been prepared for the creation of individualized therapy partially based on genetic features of the underlying disease. We provide an update on selected issues of development in this fast evolving field; however, we do not claim completeness.

Description: In a proportion of patients with chronic myeloid leukemia (CML) being treated with dasatinib, we recently observed large granular lymphocyte (LGL) expansions carrying clonal T-cell receptor (TCR) γ/δ gene rearrangements. To assess the prevalence and role of clonal lymphocytes in CML, we collected samples from patients (n = 34) at the time of diagnosis and during imatinib and dasatinib therapies and analyzed lymphocyte clonality with a sensitive polymerase chain reaction–based method of TCR γ and δ genes. Surprisingly, at CML diagnosis, 15 of 18 patients (83%) had a sizeable clonal, BCR-ABL1 negative lymphocyte population, which was uncommon in healthy persons (1 of 12; 8%). The same clone persisted at low levels in most imatinib-treated patients. In contrast, in a distinct population of dasatinib-treated patients, the diagnostic phase clone markedly expanded, resulting in absolute lymphocytosis in blood. Most patients with LGL expansions (90%) had TCR δ rearrangements, which were uncommon in patients without an LGL expansion (10%). The TCR δ clones were confined to γδ+ T- or natural killer–cell compartments and the TCR γ clones to CD4+/CD8+ αβ+ fractions. The functional importance of clonal lymphocytes as a part of leukemia immune surveillance and the putative anergy-reversing role of dasatinib require further evaluation.

Description: Opportunistic infections have an major impact on morbidity and mortality after peripheral blood progenitor cell transplantation, and are caused by deficient T cell numbers and activities, which correlate with thymic dysfunction. Keratinocyte growth factor (KGF) has been shown to protect thymic epithelial cells from injury induced by chemotherapy and radiation in mice, associated with improved thymopoiesis. We for the first time evaluated the effect of KGF on immune reconstitution after myeloablative total body irradiation (TBI) and autologous transplantation in non-human primates, a clinically relevant large animal model. Four animals received KGF 250mg/kg body weight given once (day −5) before TBI (low dose KGF) and CD34+ purified PBPC transplantation, and two animals received the same dose given 3 times (day −5, −4, −3) before and 3 times (day +1, +2, +3) after TBI (day −2, −1) and transplantation (day 0) (high-dose KGF). These KGF-treated animals were compared to an age and CD34+ cell dose-matched control group of five monkeys transplanted with purified CD34+ cells but without KGF treatment. We evaluated the reconstitution of T and B cells in blood and lymph nodes (LN) for a year post transplantation. All animals engrafted (neutrophils 〉500/ml) day by day 11 post transplantation. The animals treated with KGF had higher frequencies of naïve T cells in blood and in peripheral LN post transplantation as compared to the control animals, for instance at one month after transplantation the percentage of LN naïve CD4+ T-cells was 35±19% in the high dose KGF group, 14±2% in the low dose KGF group and 15±4% in the control group. At 3 months after transplantation the percentage of naïve CD4+ in LN was 56±5% in the high dose and 59±9% in the low dose KGF groups versus 27±7% in the control group. We observed similar findings in the CD8+ subset. All KGF treated animals had higher peripheral blood TREC levels, broader TCR repertoires as determined by spectratyping of the CDR3 region, and lower frequencies of Ki-67+ proliferating T cells, presumably due to a lower drive for peripheral expansion in animals with better thymic output of naïve T cells. The recovery of circulating B-cells was not altered with KGF treatment compared to the controls. At 12 months after transplantation there was uniform preservation of thymic architecture with proliferating thymocytes in the animals treated with KGF, in comparison to the control group of animals, with thymic atrophy seen in the majority. Thus, peri-TBI treatment with KGF resulted in protection of thymic epithelial cells, improved thymic repopulation, and increased naïve T-cell numbers in secondary lymphatic tissues, and correlated with better immune responses to newly induced (HIV envelope) as well as recall antigens including measles and CMV. KGF-mediated protection and preservation of thymic epithelial cells from TBI-mediated damage, with improved output of naïve T cells post-transplantation may improve immune reconstitution and thus outcomes in patients undergoing autologous or allogeneic transplantation.

Description: Abstract 1123 Poster Board I-145 Introduction Immunosuppressive effects of the second generation tyrosine kinase inhibitor dasatinib (Sprycel®) on T cells and NK cells have been described in vitro. In contrast, in some CML or Ph+ ALL patients receiving dasatinib, the development of a chronic, oscillating lymphocytosis has been observed in vivo. We previously showed that dasatinib-induced lymphocytosis typically comprises of oligoclonal NK or CD8+ cytotoxic T cell large granular lymphocyte (LGL) expansions and was associated with HLA-A*0201, CMV reactivation and enhanced, long-lasting therapy responses in patients with advanced leukemia. Patients and Methods To further elucidate the mechanisms underlying LGL expansions during dasatinib, we analyzed T cell and NK cell effector functions directly ex vivo. Specifically, we evaluated activation, proliferation, cytotoxic activity, cytokine secretion, degranulation, KIR expression profile and apoptosis/necrosis susceptibility. Results We observed decreased NK cell cytotoxic activity against the cell line K562 only in patients with NK cell expansions, potentially due to exhaustion based on excessive in vivo proliferation. This reduced lytic ability was restored after pre-treatment with IL-2 for 18h. No correlation between functionality and KIR expression profile was found. Furthermore, patients with LGL expansions exhibited elevated IP10/RANTES levels in the plasma compared to non-LGL patients and healthy controls. Strikingly, within the CD8+ T cell population specific for the CMV epitope NLVPMVATV (pp65, residues 495-503) restricted by HLA-A*0201, we observed distinct CD8high and CD8low fractions in patients with LGL expansions (n=5) but not in patients without LGL expansions (n=4) or in healthy controls (n=3). The CD8high subpopulation was more resistant to inhibition by dasatinib compared to other cell fractions; in addition, dasatinib effectively abrogated down-regulation of both TCR and CD8 in the CD8high subpopulation in vitro. Conclusions Thus, we hypothesize that CMV reactivation is intimately linked to the observed LGL expansions in dasatinib-treated patients. The expanded cytotoxic cells may target both CMV-infected and leukemic cells by virtue of epitope sharing and thus explain the enhanced anti-leukemic control we observed. Further investigations based on the application of polychromatic flow cytometry and analysis of T cell clonality are underway to establish the nature of this association in more detail. Dasatinib and derivatives may prove to be useful in modulating anti-leukemic/anti-host immune responses in vivo. Disclosures Mustjoki: BMS: Honoraria. Ekblom:BMS: Honoraria. Porkka:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Seggewiss:BMS: Honoraria.

Description: Dasatinib (Sprycel®, Bristol-Myers Squibb) is a dual BCR-ABL/SRC kinase inhibitor. In 2006, it was approved for the treatment of imatinib-refractory/-resistant CML and Ph+ ALL. An increased infection rate has been described in patients undergoing dasatinib treatment; we postulated that this might be due to inhibition of the SRC kinases LCK and FYN, which play an important role in T cell signalling following antigen recognition. We evaluated dasatinib’s immunobiological effects on purified human CD3+ T cells from healthy blood donors using the promiscuous tyrosine kinase inhibitor staurosporine as a comparator; we also used antigen-specific T cell clones to dissect early T cell signaling events. All assays were performed at clinically relevant doses of dasatinib (1–100nM). In CFSE dilution assays, a dose-dependent inhibition of T cell proliferation was detected with dasatinib (IC50=11nM) and staurosporine (IC50=5nM). These effects of dasatinib, but not staurosporine, were reversible; thus, T cells that were pre-incubated with dasatinib for 24h and then washed, proliferated as well as untreated T cells in the absence of dasatinib. Furthermore, we observed significant inhibition of OKT3-induced up-regulation of the activation marker CD69 (dasatinib IC50=11nM, staurosporine IC50=5nM) and reduced IL-2 production (IC50=2nM, measured by ELISA, for both dasatinib and staurosporine) in purified T cells treated with dasatinib and staurosporine. CD4+ T cells were more sensitive than CD8+ T cells to the inhibitory effects of dasatinib on activation and proliferation (activation IC50=10nM for CD4+ and 15nM for CD8+ T cells; proliferation IC50=10nM for CD4+ and 13nM for CD8+ T cells) while no differences in sensitivity were observed for staurosporine (IC50=4–5 for activation and proliferation in both CD4+ and CD8+ T cells). Since CD8+ T cell-mediated immunity is essential for long-term control of persistent DNA viruses, we evaluated dasatinib’s impact on antigen-specific alpha/beta CD8+ T cell populations specific for CMV and EBV. Profound inhibition of proliferation, cytokine secretion and degranulation was observed in all cases; these observations potentially explain the increased frequency of viral infections in dasatinib-treated patients beyond the induction of myelosuppression. Of note, gamma/delta T cell functions were also inhibited by dasatinib. Dasatinib resulted in significant upregulation of TCR/CD8 at the T cell surface of EBV specific T cell clones indicating that the drug blocks TCR downregulation and recycling at the T cell surface and therefore exerts its effects by blockade of proximal signalling pathways. Overall, these findings provide a rationale to explore the potential of dasatinib as an immunosuppressant in the settings of transplantation and T cell driven autoimmune diseases.

Description: An understanding of the number and contribution of individual pluripotent hematopoietic stem cells (HSCs) to the formation of blood lineages has important clinical implications for gene therapy and stem cell transplantation. We have been able to efficiently mark rhesus macaque long-term repopulating stem and progenitor cells with retroviral vectors, and track their in vivo contributions to hematopoiesis using the linear amplification mediated–polymerase chain reaction (LAM-PCR) technique of insertion site analysis. We assessed the impact of busulfan on contributions of individual retrovirally marked clones to hematopoiesis. There were 2 macaques that received transplants of retrovirally transduced CD34+ cells 2 years previously that were then treated with 4 mg/kg busulfan. Despite only transient and mild suppression of peripheral blood counts, the numbers of individual stem/progenitor clones contributing to granulocyte production decreased dramatically, by 80% in the first monkey and by 60% in the second monkey. A similar impact was seen on clones contributing to T cells. The clone numbers recovered gradually back toward baseline by 5 months following busulfan in the first monkey and by 3 months in the second monkey, and have remained stable for more than one year in both animals. Tracking of individual clones with insertion-site–specific primers suggested that clones contributing to hematopoiesis prior to busulfan accounted for the majority of this recovery, but that some previously undetected clones began to contribute during this recovery phase. These results indicate that even low-dose busulfan significantly affects stem and progenitor cell dynamics. The clonal diversity of hematopoiesis was significantly decreased after even a single, clinically well-tolerated dose of busulfan, with slow but almost complete recovery over the next several months, suggesting that true long-term repopulating stem cells were not permanently deleted. However, the prolonged period of suppression of many clones suggests that transplanted HSCs may have a marked competitive advantage if they can engraft and proliferate during this time period, and supports the use of this agent in nonmyeloablative regimens

Description: Opportunistic infections contribute to morbidity and mortality after peripheral blood progenitor cell (PBPC) transplantation and are related to a deficient T-cell compartment. Accelerated T-cell reconstitution may therefore be clinically beneficent. Keratinocyte growth factor (KGF) has been shown to protect thymic epithelial cells in mice. Here, we evaluated immune reconstitution after autologous CD34+ PBPC transplantation in rhesus macaques conditioned with myeloablative total body irradiation in the absence or presence of single pretotal body irradiation or repeated peritransplant KGF administration. All KGF-treated animals exhibited a well-preserved thymic architecture 12 months after graft. In contrast, thymic atrophy was observed in the majority of animals in the control group. The KGF-treated animals showed higher frequencies of naive T cells in lymph nodes after transplantation compared with the control animals. The animals given repeated doses of KGF showed the highest levels of T-cell receptor excision circles (TRECs) and the lowest frequencies of Ki67+ T cells, which suggest increased thymic-dependent reconstitution in these animals. Of note, the humoral response to a T-cell–dependent neo-antigen was significantly higher in the KGF-treated animals compared with the control animals. Thus, our findings suggest that KGF may be a useful adjuvant therapy to augment T-cell reconstitution after human PBPC transplantation.